Neurodegeneration Brainstem: Correlation with Reduced Age ...Lifelong Expression of Apolipoprotein D in the Human Brainstem: Correlation with Reduced Age-Related Neurodegeneration

Lifelong Expression of Apolipoprotein D in the HumanBrainstem: Correlation with Reduced Age-RelatedNeurodegenerationAna Navarro*, Elena Méndez, Celso Diaz, Eva del Valle, Eva Martínez-Pinilla¤, Cristina Ordóñez, JorgeTolivia*

Instituto de Neurociencias del Principado de Asturias (INEUROPA), Dpto. Morfología y Biología Celular, Facultad de Biología y Medicina, Universidad deOviedo, Asturias, Spain

Abstract

The lipocalin apolipoprotein D (Apo D) is upregulated in peripheral nerves following injury and in regions of thecentral nervous system, such as the cerebral cortex, hippocampus, and cerebellum, during aging and progression ofcertain neurological diseases. In contrast, few studies have examined Apo D expression in the brainstem, a regionnecessary for survival and generally less prone to age-related degeneration. We measured Apo D expression inwhole human brainstem lysates by slot-blot and at higher spatial resolution by quantitative immunohistochemistry ineleven brainstem nuclei (the 4 nuclei of the vestibular nuclear complex, inferior olive, hypoglossal nucleus,oculomotor nucleus, facial motor nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, andRoller`s nucleus). In contrast to cortex, hippocampus, and cerebellum, apolipoprotein D was highly expressed inbrainstem tissue from subjects (N = 26, 32−96 years of age) with no history of neurological disease, and expressionshowed little variation with age. Expression was significantly stronger in somatomotor nuclei (hypoglossal,oculomotor, facial) than visceromotor or sensory nuclei. Both neurons and glia expressed Apo D, particularly neuronswith larger somata and glia in the periphery of these brainstem centers. Immunostaining was strongest in theneuronal perinuclear region and absent in the nucleus. We propose that strong brainstem expression of Apo Dthroughout adult life contributes to resistance against neurodegenerative disease and age-related degeneration,possibly by preventing oxidative stress and ensuing lipid peroxidation.

Citation: Navarro A, Méndez E, Diaz C, del Valle E, Martínez-Pinilla E, et al. (2013) Lifelong Expression of Apolipoprotein D in the Human Brainstem:Correlation with Reduced Age-Related Neurodegeneration. PLoS ONE 8(10): e77852. doi:10.1371/journal.pone.0077852

Editor: Renping Zhou, Rutgers University, United States of AmericaReceived June 20, 2013; Accepted September 4, 2013; Published October 22, 2013Copyright: © 2013 Tolivia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by MEC (Ministerio de Educación y Ciencia) and FEDER (Fondo Europeo de Desarrollo Regional)(SAF2007-64076/)grant and Principado de Asturias (SV-PA-13-ECOEMP-80). CO was a predoctoral fellow from “Gobierno del Principado de Asturias” Spain (BP05-064). EMwas predoctoral fellows from “Ministerio de Educación y Ciencia” Spain (AP-2005-3815). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.* E-mail: [email protected] (EM); [email protected] (AN)

¤ Current address: CIMA Área de Neurociencias, Pamplona, Navarra, Spain.

Introduction

Apolipoprotein D is a glycoprotein first isolated from theplasma HDL fraction and also found in smaller amounts inVLDL and LDL. The amino acid sequence shows a highhomology with the lipocalin family but not with otherapolipoproteins. The main role of Apo D is still uncertain, but byanalogy with other lipocalins it may serve as a transporter ofsmall hydrophobic molecules between the circulation andtissue [1]. The gene for human Apo D (at 3p14.2) was clonedand sequenced in 1986, and mRNA expression subsequentlydemonstrated in many tissues [2].

The Apo D protein has since been detected in many organs,tissues, and body fluids of both humans and other species [3],

and is probably able to interact with a variety of differentligands depending location and physiological requirements.Adrenal gland and kidney express the highest levels of Apo DmRNA, and expression is also intense in pancreas, placenta,spleen, lung, ovary, testis, brain, and peripheral nerves.Lachrymal secretions, axillary apocrine gland secretions, urine,cerebrospinal fluid (CSF), and perilymph and middle ear fluidalso contain Apo D [3]. At the cellular level, Apo D is commonlyexpressed by fibroblasts, especially those close to vessels [4].

In the human peripheral nervous system (PNS), Apo D issynthesized and secreted by fibroblasts and Schwann cellsunder basal conditions and upregulated during regenerativeprocesses that follow injury. Following sciatic nerve injury, ApoD mRNA and protein expression levels increase to levels much

PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e77852

higher than all other apolipoproteins [5,6]. Like Apo E, Apo D issynthesized locally, while Apo AI and Apo A-IV are derivedfrom the bloodstream [5]. Peripheral nerve injury isaccompanied by the release of hydrophilic molecules initiallydeposited in Schwann cells, macrophages, and neurilemmalcells. Cholesterol, together with other lipids, is then used forrestructuring and membrane biogenesis. Apolipoproteinscontribute to the mobilization of lipids and cholesterolhomeostasis and have complementary roles in nerve injury inthe absence of other apolipoproteins [7].

The human central nervous system (CNS) expresses aunique Apo D isoform with lower molecular weight (29 kD) thanthat founded in CSF or plasma (32 kD). In humans and othermammals, immunohistochemical and hybridohistochemicaltechniques have demonstrated Apo D on the surfaces of glialcells (astrocytes, oligodendrocytes, and microglia), pial cells,perivascular cells, and neurons [4,8–13].

Apolipoprotein D expression is more intense in white matterthan grey matter due to particularly strong expression inoligodendrocytes, where it may be involved in myelination [7].On the contrary, expression is lower in neurons and astrocytesand more variable across brain regions [8,9]. Furthermore, thesynthesis of Apo D by astrocytes is elevated by activation inresponse to injury, termed reactive astrogliosis [10]. The factthat Apo D localizes to ependimocytes, glial cells, neuropil, andperivascular cells suggests that it could be a bidirectionalcarrier of small molecules through the blood-brain barrier (BBB)and (or) blood-CSF barrier (BCB) [8,9,14].

Increased expression of Apo D in the CNS has beenobserved in neurodegenerative and neuropsychiatric disorders(for review see 15,16). In some of these diseases, such asAlzheimer's disease (AD) and multiple sclerosis (MS), elevatedApo D expression has also been observed in the cerebrospinalfluid and plasma [17,18]. Furthermore, the correlation betweenregional Apo D expression pattern and sensitivity to neuralpathology associated with these disorders suggests that Apo Dis a marker for brain structures prone to neuropathology, suchas the frontal cortex, entorhinal cortex, and hippocampus inAD, pre-frontal cortex in schizophrenia, and temporal cortex inbipolar disorder [17,19,20]. There has been little work on Apo Dexpression in mid- or hindbrain structures such as the ponsand medulla, regions with distinct anatomical structures andrudimentary functions essential for life.

In this study, we examined the expression of Apo D inbrainstem extracts by slot-blot and in fixed sections byimmunohistochemistry. Tissues were obtain from subjects of abroad range of ages to examine age-related expressionchanges in both whole brainstem and 11 well defined nuclei ofthe human medulla oblongata and pons.

Materials and Methods

1. Human tissuesUse of human brain tissues were approved by “Comité Ético

de investigación Clínica Regional del Principado de Asturias”as follows. These studies were granted waivers of consent onthe following bases: 1) samples were gathered retrospectivelyfrom pathology archives of necropsies performed for diagnostic

purposes; 2) patient identities were anonymized andcompletely delinked from unique identifiers; and 3) there wasno risk to the participants.

All human brain tissue samples were provided by theDepartment of Pathologic Anatomy of the Central Hospital ofAsturias and obtained from necropsies within 6 h of death.These samples were the same as those used in previousstudies by our group [8-10,21]. Twenty-six brainstems frommen 32−92 years old were examined. None of the patientspresented a previous inner ear or neurological disease. In allcases, macroscopic and microscopic examination revealed theabsence of acute, chronic, localized, or diffuse brain pathology.Tissue blocks were cut into 1 cm slices perpendicular to thebrainstem axis. Two samples were obtained from each case;one was frozen at -80 ° C for protein extraction and the otherfixed in paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4)for neuropathological diagnosis and immunohistochemicalstudies. Due to the structure of the nuclei studied, samples forslot blots were not separated into white and grey mattersections. The present study was conducted according to theDeclaration of Helsinki and was approved by the “Comité Éticode investigación Clínica Regional del Principado de Asturias”(Spain).

2. Protein slot-blottingApo D levels were quantified using a slot blot technique [9].

Samples from human breast cyst fluid were also included as apositive control for Apo D detection, and ExtrAvidin-AlkalinePhosphatase (Sigma, E26366) was used as an internal control.Briefly, 0.06, 0.12, 0.25, 0.5, 1.0, and 2.0 μg samples of totalprotein were slot blotted under vacuum in a Bio Dot apparatus(Biorad). Nitrocellulose membranes were then immunostainedfor Apo D protein according to the following protocol. Non-specific binding was blocked by incubation of membranes with1% bovine serum albumin (BSA). Blocked membranes werethen incubated for 1 hour at room temperature with a primaryantibody against human Apo D (1:10,000; a gift from Dr. CarlosLópez Otín, Departamento de Bioquímica y Biología Molecular,Universidad de Oviedo) [8,22-24]. After 3 washes of 15 mineach in PBS, membranes were incubated for 30 min at roomtemperature in biotinylated monoclonal anti-rabbit IgG(1:10,000; Sigma, B 5283), washed again as before, and thenincubated with ExtrAvidin Alkaline Phosphatase (1:10,000;Sigma, E26366). After three washes of 15 min each in PBS,enzyme activity was measured by incubation in Sigma FastBCIP/NBT solution (Sigma, B5655) for 2 hours at roomtemperature.

The blots of Apo D were dried and bands quantified asrelative optical densities (arbitrary units) using a digital scanner(Nikon AX-110, Nikon) and ImageJ 1.37c (National Institutes ofHealth, Bethesda, MD, USA) [9].

3. Cytoarchitectonic staining andImmunohistochemistry

After fixation, tissue blocks were washed in distilled water,dehydrated, cleared in butyl acetate, and embedded in paraffin.Sections were cut at 10 μm thickness and mounted on"SuperFrost Plus" (Mentzel-Glasse) slides, dried at 36 °C for

Apo D Expression in Human Brainstem


24 h, deparaffined in xylene, and partially rehydrated byincubation in a graded alcohol series (2 min per concentration).In each case, every 80th or 160th section was stained by theNissl/Myelin technique [25] and examined formyelocytoarchitecture. The brainstem nuclei examined werethe principal inferior olivary nucleus (IO), the hypoglossalnucleus (XII), the oculomotor nucleus (III), the facial nucleus(VII), the Roller nucleus (RN), the dorsal motor nucleus of thevagus nerve (DX), nucleus of the solitary tract (NST), inferiorvestibular nucleus (IVN), medial vestibular nucleus (MVN),lateral vestibular nucleus (LVN), and superior vestibularnucleus (SVN). The boundaries of these nuclei were definedaccording to [26] as in previous studies by our group [27,28].

For immunohistochemistry, fixed sections were permeablizedwith triton X (0.1%, 5 min), washed in distilled water, treatedwith H2O2 (3%, 5 min) to quench endogenous peroxidaseactivity, washed in distilled water, and treated with PBS. Afterblocking non-specific binding by incubation in BSA (30 min),sections were incubated with the antibody against human ApoD (1:2,000) overnight at 4 °C. Immunoreactivity was detectedusing the Extravidin biotin peroxidase staining kit (Sigma Extra3), and peroxidase activity visualized by incubation with SigmaFast DAB (Sigma D4168) at room temperature for 30 min.Finally, the sections were counterstained using a modifiedformaldehyde thionin method [29], dehydrated, cleared ineucalyptol, and mounted with Eukitt. For controls,representative sections were processed in the same way with anon-immune serum or specifically absorbed sera in place of theprimary antibody. Under these conditions, no specificimmunostaining was observed.

4. Quantification of Apo D ExpressionFor quantification of Apo D expression, the chromogenic

signal was selected using Adobe Photoshop CS 8.0.1 (AdobeSystems Inc., CA, USA) and density measured by ImageJ1.37c (National Institutes of Health, Bethesda, MD, USA)according to a procedure developed by our group [30]. Briefly,sections were photographed with a digital camera and theimages imported into Adobe Photoshop. For subsequentcomparison of Apo D expression in different sections, themagnification of images must also be equal. The analysisprotocol is as follows: (1) To select the specific chromogenicsignal, choose “Color range” in the “Select menu” ofPhotoshop, and with the “Eyedropper” tool click on any objectin the image displaying the desired color/chromogen and allareas within the selected color range will be highlighted in anautomatically generated clone image. The tolerance bar of thecolor range tool makes direct visualization of the selectedregions possible with minimal variations in the chromogenselection. Once chromogen selection is finished, the profileused can be archived for use with similar stained sections (2).Close the “Color range” panel and all immunopositive objects inthe original image appear highlighted (3). Open the “Edit” menuof Photoshop, select “Copy”, open a “New file”, and “Paste” theselected image. The new image shows only the positiveselected areas on a white background (4). Convert the imageto greyscale so all selected marked areas are represented ingrey tones correlated with chromogen intensity. This greyscale

picture must be saved in ‘tif’ format, which can then be openedin Scion Image (or other free image analysis programs such asImageJ) (5). Open the picture in this program and calculate the“Mean density” under “Uncalibrated” conditions. Themeasurements obtained show the signal strength and can becopied and exported to a spreadsheet (i.e., Excel). The signalstrength varies between 0 and 255 (value 0 corresponds to anonstained section (white), and 255 corresponds to a fullystained section (black).

5. Statistical analysesAll statistical calculations were conducted using SPSS 15.0

for Windows. Data sets were first tested for normality using theKolmogorov-Smirnov test with Lilliefors correction. Expressionlevels of Apo D were compared among nuclei by the Mann-Whitney U test due to non-homogeneity of variance. A P value< 0.05 was considered statistically significant.

Results

1. Apo D protein levelsThe levels of Apo D expression were measured in 26 human

brainstem samples from patients ranging in age from 32 to 96years. Densitometric analysis of slot blots showed robust andrelatively stable expression throughout adult life, particularlybetween the forth and eighth decades (Figure 1). Regressionanalysis showed no statistically significant increase inexpression with age.

2. Tissue distribution of Apo D proteinExpression of Apo D was also measured at the cellular level

in 11 pontine and medullar nuclei of both sensorial and motorfunctional systems (Figure 2). All special somatosensory nucleistudied are components of the vestibular nuclear complex(VNC). In addition, we assessed Apo D expression in theinferior olivary nucleus (IO), which does not belong to either thegeneral or special somatosensory system but hassomatosensory functions (Figure 2). We also studied thehypoglossal nucleus (XII), oculomotor nucleus (III) and thefacial motor nucleus (VII) as examples of somatomotor andbranchimotor nuclei. The nucleus of the solitary tract (NST) andthe dorsal motor nucleus of the vagus nerve (DX) were studiedas examples of visceromotor nuclei. Finally, we studied a smallmotor nucleus located in the middle reticular substance namedRoller`s nucleus (RN).

2.1. Somatomotor and branchimotor system nuclei. Allsomatomotor and branchimotor system nuclei studied showedrobust Apo D immunoreactivity throughout life. The hypoglossalnucleus (XII), located in the middle and lower third of themedulla oblongata, is part of the somatic motor column of thebrainstem. This somatomotor nucleus (Figure 3A, B) exhibitedthe highest level of Apo D staining after the facial nucleus(Figure 3E, F). In XII, protein expression was not evenlydistributed but rather located principally in larger neurons(Figure 3B). There was no significant relationship between ApoD expression and age, but there was a modest tendency for



total protein expression to diminish in older subjects (r =-0.5239; p > 0.05).

The oculomotor or abducens nucleus (III) is also part of thesomatic motor column of the brainstem and shares many

characteristics with XII (Figure 3C, D). The III also exhibitedintense Apo D immunoreactivity, but expression washeterogeneous, with the middle third showing the greatestprotein expression, coinciding with the highest cell density.

Figure 1. Slot blot analysis and amount of Apo D protein along different ages and decades. A) Samples from medullaoblongata of different subjects during aging. B) Amount of Apo D in samples from subjects between 35 and 82 years old. Thegraphic shows the densitometric analysis of slot blots. Data presented as means of relative optical density (ROD). Each point in thegraph represents mean density ±standar error of the mean. Regresion lines and Pearson`s correlation coefficient (r) is also shown.doi: 10.1371/journal.pone.0077852.g001



Figure 2. Hemisections of medulla oblongata. Four levels of medulla oblongata (A,B,C,D), stained with a Nissl/Myelintechnique, with the localization of the different nuclei studied in the present paper are showed.doi: 10.1371/journal.pone.0077852.g002



Figure 3. Immunohistochemistry for Apo D and amount of Apo D protein in some nuclei along different decades. A, B)Hypoglossal nucleus (XII). C,D) Oculomotor nucleus (III). E, F) Facial motor nucleus (VII). Roller`s nucleus (RN). Data presented asmeans of relative optical density (ROD). Each point in the graph represents mean density in a x20 field ±standar error of the mean.Regresion lines and Pearson`s correlation coefficient (r) are also shown. Bar: 40 μm.doi: 10.1371/journal.pone.0077852.g003



There was no significant relationship between Apo Dexpression and age, but there was a slight tendency for totalprotein expression to increase (r = 0.2098; p > 0.05). As in theother nuclei studied, Apo D was present in almost all neuronsof medium and large size.

The facial nucleus (VII), a nucleus with well describedtopographic organization, contained many large Apo D-positiveneurons and showed the highest Apo D expression of all nucleistudied (Figure 3E, F). The density of stained cells was highestin the middle third of the nucleus, decreasing in the rostral andcaudal ends. There was no change in Apo D expression withage (r = -0.0329; p > 0.05; Figure 3E).

The immunoreactivity of Roller’s nucleus (Figure 3G, H) wasalso very high despite being smaller than the other nucleistudied (Figure 3G, H). We found no significant relationshipbetween age and total Apo D expression in RN, but there wasa slight tendency to increase with age (r = 0.5404; p > 0.05).

2.2. Viscerosensory and visceromotor systemnuclei. The DX (Figure 4A, B) and NST (Figure 4C, D) werestudied as examples of visceromotor nuclei. Both exhibited lowintensity immunostaining compared to the somatomotor andbranchiomotor nuclei, and showed no statistically significantchanges with age (r = 0.0059; r = 0.2981; p > 0.05). In general,these low levels of Apo D expression mirrored other sensorynuclei (Figure 5), with all showing lower immunoreactivity thansomatomotor nuclei (Figure 6).

2.3. The inferior olive and somatosensory systemnuclei. The general and special somatosensory nuclei of thebrainstem are located in the pons and medulla oblongata. Wealso studied IO (Figure 4E, F) due to its somatosensoryfunctions and because the cytoarchitectural features can beeasily delimited. The mean densitometric value of Apo Dexpression in the IO was intermediate between the motornuclei (with generally high expression) and the sensory nuclei(with generally low expression). There was no statisticallysignificant relationship between the densitometric value of ApoD staining and age, but there was a small tendency forincreased expression with age (r = 0. 1377; p > 0.05).

The VNC consists of inferior (IVN), lateral (LVN), medial(MVN), and superior (SVN) components. The IVN containsneurons of a broad range of sizes, but medium-size neuronsare predominant (Figure 5A, B). The IVN and LVN had thehighest levels of Apo D expression in the VNC. In the IVN, ApoD expression was located only in the cytoplasm of largerneurons. No statistically significant relationship between ageand Apo D expression was found in the IVN, although therewas a modest trend for a decrease with age (r = -0.414; p >0.05). The MVN is the largest nuclei of the VNC and denselypacked with intermediate-size neurons. Despite the high celldensity, Apo D expression was relatively low. Staining waspreferentially located in neurons of greatest size (Figure 5C,D). Densitometric analysis revealed no significant changes inexpression with age (r = 0.0033; p > 0.05). The LVN exhibitedthe highest Apo D expression in the VNC but again with nosignificant age-related changes (r = -0.2228; p > 0.05). In theLVN, all giant neurons (Deiters’ cells) showed high levels ofApo D expression regardless of subject age (Figure 5E, F). TheSVN consists of large neurons arranged in nests among

myelinated fibres that cross the nucleus as well as smallerisolated neurons (Figure 5G, H). The SVN expressed the leastamount of Apo D of all VNC components and also showed nostatistically significant changed in expression with age (r =-0.2458; p > 0.05).

Unlike forebrain regions and cerebellum, we found intenseApo D expression in many neurons and glial cells in younghuman brainstem nuclei. Immunostaining for Apo D wasusually restricted to the neuronal cytoplasm, with highestintensity in the perinuclear region. When the signal was strong,it tended to fill the entire cell, even the neurites, but has beennot observed in the cell nucleus. In general, larger neuronsshowed more intense staining for Apo D than smaller neurons.The number of Apo D-positive glial cells appeared highest atthe periphery compared to the core of these nuclei (Figures3-5). Finally, motor nuclei exhibited higher Apo D expressionthan sensory nuclei (p < 0.006). Again, expression level wasindependent of age (Figure 6). The physiological implications ofthese observations required further study.

Discussion

In the present study, we found that expression of Apo D inmedulla oblongata and pons is already high in young subjects,contrary to other areas of the brain previously studied [8,9], andwidely distributed among the different neuronal nuclei in thisregion. We examined expression at the cellular level in elevensuch nuclei and found robust expression in neurons, glial cells,and neuropil even in young brainstem. This is also in contrastto human cerebral cortex, where little Apo D is localized toneurons and appears in only a few astrocytes [8,9]. The region-specific distribution of Apo D expression was first studied indetail by our group [8] and later reported by others [19,20]. Wepreviously demonstrated differences in Apo D expressionamong neuronal nuclei located in the same brain region [24] oramong neurons of the same area [23]. Moreover, the absenceof Apo D expression appears to enhance neuronal vulnerabilityto death [23,24]. Thus, we consider these results significant forfuture pathological studies on brainstem in various neurologicaldiseases. The pattern of Apo D expression in human braincoincides with other lipocalins that are lipopolysaccharide(LPS)-inducible [31,32]. The highest level of lipocalin2 (LCN2)was found in the olfactory bulb, followed by the brainstem,cerebellum, hypothalamus, and thalamus, with lowest levels inthe striatum, hippocampus, frontal cortex, and somatosensorycortex of normal mouse brain [33].

In human frontal cortex, expression is higher in white matterthan grey matter due to higher synthesis by oligodendrocytes[8,9,13,34]. Whether this is also true in brainstem remains to bedetermined as the complex distribution of nuclei and fibrebundles does not allow for easy separation of white and greymatter for lysate preparation. Nonetheless, we noted the mostintense expression in neurons, particularly larger neurons ofsomatomotor nuclei.

Increased expression of Apo D in the CNS has beenassociated with aging, neurodegenerative diseases, andneuropsychiatric disorders. However, the majority of studies onApo D have focused on cerebral and cerebellar cortices and



the hippocampus, areas particularly prone to pathologicalchanges with age and during the progression of

neurodegenerative diseases. In all these areas, Apo Dexpression in young subjects was lower in grey matter than

Figure 4. Immunohistochemistry for Apo D and amount of Apo D protein in some nuclei along different decades. A, B)Dorsal motor nucleus of the vagus (DX). C,D) Nucleus of solitary tractis (NST). E, F) Inferior olivary nucleus (IO). Data presented asmeans of relative optical density (ROD). Each point in the graph represents mean density in a x20 field ±standar error of the mean.Regresion lines and Pearson`s correlation coefficient (r) are also shown. Bar: 40 μm.doi: 10.1371/journal.pone.0077852.g004



Figure 5. Immunohistochemistry for Apo D and amount of Apo D protein in vestibular nuclear complex along differentdecades. A, B) Inferior vestibular nucleus (IVN). C,D) Medial vestibular nucleus (MVN). E, F) Lateral vestibular nucleus (LVN). G,H) Superior vestibular nucleus (SVN). Data presented as means of relative optical density (ROD). Each point in the graphrepresents mean density in a x20 field ±standar error of the mean. Regresion lines and Pearson`s correlation coefficient (r) are alsoshown. Bar: 40 μm.doi: 10.1371/journal.pone.0077852.g005



white matter, and increased in both tissues with age andfollowing injury. Apo D expression has also been linked tocellular senescence and growth arrest in cell culture [13,35],and expression appears correlated with age in tissues asdiverse as hair follicles and the CNS [14]. Even a previousstudy by our group found that Apo D protein and mRNA

increased during normal development and aging in frontalcortex [9]. Moreover, a similar age-dependent expression wasdemonstrated in the frontal cortex of rats and monkeys [36,37].By contrast, slot blots and densitometric quantification of Apo Dexpression revealed that Apo D expression in brainstem isalready high in young to middle-age adults with no history of

Figure 6. Mean of Apo D protein during aging in each nuclei of medulla oblongata studied. Nuclei are grouped accordingtheir sensory or motor function. Motor nuclei show higher quantity of Apo D than sensory nuclei. Data presented as means ofrelative optical density (ROD). Bars represent mean value of Apo D along all decades.doi: 10.1371/journal.pone.0077852.g006



neurodegenerative disease or brain injury. Moreover,expression was relatively stable during aging, a resultinconsistent with current assumptions that Apo D is an “age-related” protein.

The underlying molecular mechanisms of CNS aging includeinstability of nuclear and mitochondrial genomes,neuroendocrine dysfunction, oxidative stress, altered calciummetabolism, and inflammation-mediated neuronal damage [38].A growing body of evidence points toward oxidative stresscaused by excess reactive oxygen species (ROS) productionas one of the primary determinants of age-related neuraldamage [39]. Stressors that cause an extended growth arrest,such as UV light or hydrogen peroxide, increase Apo Dexpression [32]. In fact, the promoter region of the human ApoD gene contains several stress-activated regulatory elementslike serum, acute phase, and stress response elements (SRE,APRE, or STRE) [40]. Several studies postulated that Apo Dcould have an important function in cellular defence againstage-associated oxidative stress (OS). Indeed, studies on the flyhomologue and the Apo D null mouse [41,42] showed reducedtolerance to the ROS generator paraquat (PQ) in mutant/nullgenotypes, an effect attributed to the control of lipidperoxidation. Conversely, transgenic animals overexpressinghuman Apo D [43,44], cells transfected with the gene for Apo D[20], or the direct addition of exogenous Apo D [32,45]protected against oxidants. A recent paper on PQ-treatedcerebellum from transgenic mice supports the hypothesis thatthis lipocalin is necessary for a proper CNS response tophysiological and pathological OS. The absence of Apo Dmodified the response of genes related to OS management ormyelination, while overexpression of human Apo D in neuronsalmost completely abolished the early transcriptional responseto OS [42].

We observed strong Apo D expression in all studied nuclei ofthe medulla oblongata and pons from both young and oldsubjects, and no statistically significant increase in Apo Dexpression with age. Thus, Apo D is not a marker of aging inthe brainstem. Due to these high levels of Apo D, we speculatethat human brainstem is not exposed to damaging OS duringaging, that Apo D expression in this brain region is uniquelyregulated, or that the predominant role for Apo D in this brainregion is distinct from that in other regions. It has long beenknown that Apo D is critical for remyelination of rat sciaticnerve, as evidenced by the > 40-fold increase in expression inregenerating nerve compared to non-injured control nerve [6].Elevated expression of Apo D has been found in endoneuralfibroblasts of regenerating crushed nerve in rats [6]. Since ApoD is released into the extracellular environment, Spreyer et al.[6] proposed that Apo D participates in intraneural lipidtransport and (or) re-utilization for the biosynthesis ofmembranes. In mouse peripheral nerve, Apo D is expressed bySchwann cells, by satellite cells, and by cells located betweenthe Schwann cells [34]. We demonstrated that Apo D facilitatesthe recovery of locomotor function after injury, promotes myelinclearance, and regulates the extent of angiogenesis and thenumber of macrophages invading the injury site in mousecrushed sciatic nerve. Moreover, the regeneration andremyelination of axons were delayed in the absence of Apo D

and stimulated by excess Apo D [7]. In the present study, wefound that motor nuclei have higher levels of Apo D expressionthan sensory nuclei. We argue that this may be because motornuclei require a greater capacity for myelination andmaintenance of axonal transport.

Finally, Apo D may also regulate neuritogenesis andsynaptogenesis. Co-cultures of adipocytes with peripheralneurons showed enhanced neurite and synapse formation. Ithas reported that apolipoproteins D and E3 exert neurotrophicand synaptogenic effects in dorsal root ganglion cell cultures[46]. Considering these results together, we suggest that Apo Dis critical for neural function and homeostasis. The brainstemexhibits stable expression of Apo D throughout life due to itscritical importance for survival. This brain region containscenters that regulate several functions vital to survival,including heartbeat, breathing, blood pressure, digestion,swallowing, and vomiting. It is also responsible for stimulatingthe reticular formation, a region involved in regulation of sleep/waking patterns and reflexes for maintaining muscle tone,posture, and gaze.

The effects of aging on the CNS have been studied for morethan a century, but only a few works have focused on effects tothe brainstem. We previously reported that aging is notassociated with neuronal loss in brainstem nuclei [28], only ashift from larger to smaller neurons and an increase in glial cellnumber [27]. Indeed, most studies of the brainstem have foundlittle neuronal loss and no significant functional impairment. Wepreviously described a statistically significant neuron decreasein the VNC during aging, but only in the MVN [28], which couldaccount for the small deficits in balance observed in olderindividuals. One interesting result of the present study was thatthe MNV, the only nucleus of the VNC that loses neuronsduring aging, had the lowest Apo D expression of all VNCnuclei. That basal Apo D expression is higher in brainstem thanin grey matter areas that lose neurons during aging and aregenerally more sensitive to oxidative stress [8,21,23,24]strongly suggests that the preservation of brainstem nucleiduring aging is directly related to stable expression of Apo D.

Conclusions

Although the full spectrum of Apo D functions is unclear, thecurrent study strongly suggests that this molecule is critical forresistance to age-related neural damage. Neurons that do notsynthesize or import Apo D from surrounding glial cells aremore vulnerable to age- and disease-related death. Diseasesor injuries of the brainstem region are normally incompatiblewith life, requiring stable basal expression of Apo D (andpossibly other neuroprotective factors). The results of this studysupport the hypothesis that Apo D is a critical neuroprotectivemolecule against neurodegenerative disease and the ravagesof age.

Author Contributions

Conceived and designed the experiments: AN EM CD EV EMPCO JT. Performed the experiments: EM CD EV EMP CO.Analyzed the data: AN EM CD JT. Contributed reagents/



materials/analysis tools: EM CD EV EMP CO. Wrote themanuscript: AN EV EMP JT.

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Lifelong Expression of Apolipoprotein D in the Human Brainstem: Correlation with Reduced Age-Related NeurodegenerationIntroductionMaterials and Methods1. Human tissues2. Protein slot-blotting3. Cytoarchitectonic staining and Immunohistochemistry4. Quantification of Apo D Expression5. Statistical analyses

Results1. Apo D protein levels2. Tissue distribution of Apo D protein

DiscussionConclusionsAuthor ContributionsReferences

Documents

Neurodegeneration Brainstem: Correlation with Reduced Age ...Lifelong Expression of Apolipoprotein D in the Human Brainstem: Correlation with Reduced Age-Related Neurodegeneration